摘要
目的建立类鼻疽伯克霍尔德氏菌感染人肺癌上皮细胞A549的细胞模型。方法优化类鼻疽伯克霍尔德杆菌感染A549细胞的感染条件(如MOI、感染时间),通过活细胞工作站动态观察、Giemsa染色、激光共聚焦、透射电镜确证胞内感染和宿主细胞的形态变化,通过炎症因子IL-8和TNF-α的检测,分析病原菌入胞率和宿主反应性,评价该模型中病原菌侵入A549导致多核巨细胞(multinuclear giant cell,MNGC)形成的特点和病理损伤规律。结果透射电镜结果显示类鼻疽伯克霍尔德氏菌侵入到胞内的时间最早是4 h。通过Giemsa染色形态观察,类鼻疽伯克霍尔德氏菌感染后最早8 h可观察到典型MNGC的形成。随着感染时间的延长,MNGC形成率和炎症因子IL-8逐渐升高,而TNF-α在此模型中变化不明显。结论成功构建类鼻疽伯克霍尔德氏菌感染A549细胞模型。
Objective To establish an infection model of A549 cells by Burkholderia pseudomallei in vitro to study its pathogenesis. Methods The purpose of this study was to optimize the methods in infection of A549 cells by Burkholderia pseudomaUei, such as multiplicity of infection （MOI） and incubation time. The number of intracellular bacteria and the formation of muhinuclear giant cells （MNGCs） were visualized by Giemsa staining, live-cell digital video gallery, immunofluorescence assay and transmission electron microscopy. Furthermore, the expression of inflammatory cytokines, interleukin （IL）-8 and tumor necrosis factor （TNF）-α were detected. Results Monolayers of A549 cells were co-incubated with Burkholderia pseudomallei （ MOI = 100：1 ） and centrifugated for 5 rain at 170 x g. At 1 hour of post-infection, the extracellular bacteria were killed by the addition of 250 μg/mL kanamycin. Intracellular bacteria of A549 cells after infection with Burkholderia pseudomallei were observed as early as 4 h post-infection. MNGCs were observed in the 8 after infection, and Burkholderia pseudomallei induced release of IL-8 from A549 cells in a dose- and time-dependent fashion, whereas TNF-ot was not detected. Conclusion A burkholderia pseudomallei invaded A549 cell model is successfully established.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第19期2010-2013,共4页
Journal of Third Military Medical University
基金
全军医学科研"十二五"重大项目(AWS11J011-04)
第三军医大学重大军事预研基金(2012XJY03)~~
